Coagulation factor VIII with reduced immunogenicity

ABSTRACT

The invention describes factor VIII molecules with reduced capacity to elicit activation of NKT cells for use in the treatment of congenital and/or acquired haemophilia A and in bleeding disorders. Said factor VIII molecule is obtainable by:
         a. identification of at least one NKT cell epitope wherein said epitope comprises hydrophobic aminoacid residues in position P1 and/or P7   b. modification of said epitope(s) by eliminating at least one hydrophobic aminoacid residue in position P1 and/or P7, substituting at least one hydrophobic aminoacid residue in position P1 and/or P7 with a non-hydrophobic residue, or adding a non-hydrophobic residue in position P1 and/or P7.

INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: 1,406 bytes (Text) file named “512194_ST25.TXT” created Nov.19, 2014.

FIELD OF THE INVENTION

The present invention relates to molecules of coagulation factor VIIIwith reduced or lack of immunogenicity and their use in the therapy ofcoagulation disorders and in particular in the treatment of type Ahemophilia.

BACKGROUND OF THE INVENTION

Factor VIII is a coagulation factor acting as a co-factor in thegeneration of thombin, an essential component of coagulation. In theabsence or insufficiency of functional factor VIII, individuals sufferfrom bleeding disorders, collectively called hemophilia A. There are twotypes of hemophilia A, depending from its origin, either genetic(spontaneous hemophilia) or acquired (acquired or autoimmunehemophilia). Spontaneous hemophilia A is a disease affecting males duethe location of the factor VIII gene on the X chromosome. Women arecarriers but do not suffer from bleeding disorders because of thepresence of 2 X chromosomes. Spontaneous hemophilia is further dividedinto 3 subsets of patients, defined according to the level ofcirculating factor VIII: severe hemophilia (less than 1% factor VIII),mild hemophilia (1 to 5% factor VIII) and moderate (factor VIIIconcentrations between 5 and 10%).

Patients suffering from hemophilia require a substitution therapy byfactor VIII. This is a continuing therapy for severe hemophilia Apatients due to the increased risk of spontaneous, sometimeslife-threatening bleeding, or intermittent in mild or moderatehemophilia patients in whom factor VIII is required when there is atrauma or surgery and an acute demand of increased factor VIIIconcentrations.

By far the main complication patients suffering from hemophilia A haveto face is the emergence of antibodies towards the therapeutic agent(factor VIII) used to restore a functional coagulation.

There are currently 2 types of factor VIII used for replacement therapy,plasma-derived and recombinant. Plasma-derived factor VIII is producedfrom pools of human plasma and contains additional proteins and inparticular the physiological chaperon of factor VIII, von Willebrandfactor. Recombinant factor VIII is produced by genetic engineering andproduction by cells of animal or of human origin. Recombinant factorVIII is pure and do not contain von Willebrand factor. Vivid controversyis ongoing to decide whether there is a significant difference in therisk of eliciting an anti-factor VIII immune response when using eitherplasma-derived or recombinant factor VIII molecule. Whatever thesituation, on average 25% of patients receiving factor VIII as atherapeutic agent raise antibodies which inhibit the activity of thereplacement agent. Such antibodies are called factor VIII inhibitors.

There is no cure for factor VIII inhibitors. On empirical grounds, ithas been demonstrated that the administration of very high doses offactor VIII on a daily basis can result in some cases in a disappearanceof inhibitors. This therapy, called induction of immune tolerance, isnot reliable in its success. The lack of surrogate markers able topredict the outcome of immune tolerance de facto limits its use in anattempt to eliminate the formation of factor VIII inhibitors. Moreover,the prohibitive cost related to tolerance induction is such that only afew patients can be considered for tolerance induction.

Factor VIII inhibitors are high-affinity specific antibodies, whichimplies the participation of T lymphocytes in their formation. Theconsequence of this is that the immune response is fully memorized,leaving populations of memory B cells, which upon stimulation, transforminto plasmocytes producing antibodies, and memory T cells which maintainthe capacity to mount further antibody response upon each subsequentexposure to factor VIII. Patients presenting with factor VIII inhibitorscan not be treated with factor VIII not only because inhibitorsneutralize factor VIII function and, likely, increase the clearance rateof factor VIII, but also because each further exposure to factor VIIIincreases the concentrations of such inhibitors.

Patent application WO 2009/101206 describes a method by which it ispossible to eliminate the production of inhibitors by acting at thelevel of adaptive immunity, namely at the level of the interactionbetween factor VIII-specific T and B cells. This application describeshow the risk of producing new inhibitors upon factor exposure can beeliminated, but also how existing inhibitors can be eradicated. However,we have unexpectedly discovered that factor VIII is a very potentactivator of the innate immunity, which appears to be a pre-requisitefor eliciting an adaptive response and inhibitors. Given that factorVIII has to be administered on a regular basis (e.g. 2 or 3 times a weekfor severe hemophilia A patients), and that therefore the risk ofeliciting a new production of factor VIII inhibitors persists, there isan urgent need to define methods by which therapeutic factor VIIImolecules could be produced, which have lost their capacity to activatethe innate immunity.

The PCT application PCT/EP2011/070911 describes methods by whichproteins with capacity to activate NKT cells can be transformed so as toloose such a capacity. Thus, NKT cells are part of the innate immunesystem, which is conventionally defined as lacking memorization.However, as described in PCT application PCT/EP2011/070911 NKT cells canrecognize and be activated by the presentation of hydrophobic peptidesby the CD1d molecule. As the peptide is derived from an antigen forwhich NKT cells are specific, this represent an antigen-specific innateimmune system activation. The above-mentioned PCT application describesmethods by which proteins showing the property to activateantigen-specific NKT cells can be modified by aminoacid substitution ordeletion, thereby eliminating the capacity to bind to CD1d.

The present invention describes molecules of factor VIII obtained by themethodology described in PCT application PCT/EP2011/070911 which havelost their capacity to activate the innate immune system and,consequently, show a lack or significantly reduced capacity to active anadaptive immune response with production of inhibitors. The inventionfurther describes the use of such factor VIII molecules for thetreatment of patients in need for replacement therapy, and in particularsevere hemophilia A patients. The present invention also disclosesmethods in which gene therapy using factor VIII molecules of the presentinvention can be used.

SUMMARY OF THE INVENTION

The present invention relates to the production of molecules of factorVIII with reduced immunogenicity.

The present invention also relates to the use of said factor VIIImolecules for the treatment of patients in need for said treatment.

The PCT application PCT/EP2011/070911 describes methods to obtainpeptides or polypeptides with reduced capacity to activate NKT cells.Thus, we made the unexpected finding that a significant proportion ofpeptides or polypeptides carried aminoacid sequences which allow them tobind and to be presented by CD1d determinants for activation of naturalkiller T (NKT) cells. Activation of such cells results in release ofcytokines and, in some cases, in acquisition of, or increase incytolytic properties.

The present invention relates in one aspect to the use of at least oneisolated polypeptide used as an allofactor, which has been modified toeliminate at least one hydrophobic amino acid residue involved in theformation of an epitope recognized by NKT cells, for the manufacture ofa medicament for preventing in a subject immune responses to saidallofactor.

More specifically, the present invention relates to factor VIII and useof factor VIII as a medicament. Factor VIII is a co-factor of thecoagulation system which participates to activation of thrombin byfacilitating the formation of tenase, a serine esterase which assemblesfactor VIII, factor IX and factor X. Factor X carries the enzymaticactivity converting thrombin. In the absence of factor VIII, the rate oftenase formation is drastically reduced, leaving the patients at risk ofspontaneous bleedings, which are often life-threatening, and requiresprompt therapeutic measures. When the concentration of factor VIII ismoderately reduced (between 5 and 10%), the patients usually bleed onlyunder trauma or surgery.

Patients suffering from severe hemophilia A (less than 1% factor VIII)have frequent spontaneous bleedings and require prophylactic treatmenteither on continuous administration or bolus injection 2 or 3 times aweek. In addition, patients in whom an increased catabolism of factorVIII is observed, as in septic shock, acute fibrinolysis, polytrauma orcerebral hemorrhage, are also in need for factor VIII administration.

Definitions

The term “peptide” when used herein refers to a molecule comprising anamino acid sequence of between 2 and 200 amino acids, connected bypeptide bonds, but which can in a particular embodiment comprisenon-amino acid structures (like for example a linking organic compound).Peptides according to the invention can contain any of the conventional20 amino acids or modified versions thereof, or can containnon-naturally occurring amino acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification. The term“polypeptide” when used herein refers to generally longer peptides orproteins.

The term “epitope” when used herein refers to one or several portions(which may define a conformational epitope) of a protein which is/arespecifically recognized and bound by an antibody or a portion thereof(Fab′, Fab2′, etc.) or a receptor presented at the cell surface of a Bor T cell lymphocyte, and which is able, by said binding, to induce animmune response.

The term “antigen” when used herein refers to a structure of amacromolecule comprising one or more hapten(s) and/or comprising one ormore T cell epitopes. Typically, said macromolecule is a protein orpeptide (with or without polysaccharides) or made of proteic compositionand comprises one or more epitopes; said macromolecule can hereinalternatively be referred to as “antigenic protein” or “antigenicpeptide”.

The term “T cell epitope” or “T-cell epitope” in the context of thepresent invention refers to a dominant, sub-dominant or minor T cellepitope, i.e., a part of an antigenic protein that is specificallyrecognized and bound by a receptor at the cell surface of a Tlymphocyte. Whether an epitope is dominant, sub-dominant or minordepends on the immune reaction elicited against the epitope. Dominancedepends on the frequency at which such epitopes are recognized by Tcells and able to activate them, among all the possible T cell epitopesof a protein. In particular, a T cell epitope is an epitope bound by MHCclass I or MHC class II molecules.

The term “NKT cell epitope” refers to a part of an antigenic proteinthat is specifically recognized and bound by a receptor at the cellsurface of a T lymphocyte. In particular, a NKT cell epitope is anepitope bound by CD1d molecules.

The term “CD4+ effector cells” refers to cells belonging to theCD4-positive subset of T-cells whose function is to provide help toother cells, such as, for example B-cells. These effector cells areconventionally reported as Th cells (for T helper cells), with differentsubsets such as Th0, Th1, Th2, and Th17 cells.

The term “NKT cells” refers to cells of the innate immune systemcharacterized by the fact that they recognize epitopes presented by theCD1d molecule. In the context of the present invention, NKT cells canbelong to either the type 1 (invariant) subset (iNKT), or any othersubset which would be activated by presentation of peptidic epitopes byCD1d. The term “NKT cells” also includes NKT cells belonging to eitherthe CD4 or CD8 lineage. NKT cells often carry receptors such as NK1.1and NKG2D.

The “CD1d molecule” refers to a non-MHC derived molecule made of 3 alphachains and an anti-parallel set of beta chains arranged into a deephydrophobic groove opened on both sides and capable of presentinglipids, glycolipids or hydrophobic peptides to NKT cells.

The term “immune disorders” or “immune diseases” refers to diseaseswherein a reaction of the immune system is responsible for or sustains amalfunction or non-physiological situation in an organism. Immunedisorders in the context of the present invention refer to pathologyinduced by infectious agents and tumor surveillance.

The term “allofactor” or “alloantigen” refers to a protein, peptide orfactor (i.e. any molecule) displaying polymorphism when compared betweentwo individuals of the same species, and, more in general, any protein,peptide or factor that induces an (alloreactive) immune response in thesubject receiving the allofactor. By extension, allofactors also includegenetically-modified proteins used for feeding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of CD1d+ cells (%) plotted for indicated samples.

FIG. 2 is a graph of cell count from CD3+/CD4+ plotted for indicatedsamples.

FIG. 3A is a graph of FVII IgG antibodies (μg/ML) plotted for indicatedsamples.

FIG. 3B is a graph of FVII inhibitors (BU) plotted for indicatedsamples.

FIG. 4 is a graph of CD1d+ cells (%) plotted for indicated samples.

FIG. 5 is a graph of the number of cell clusters observed in indicatedsamples.

FIG. 6 is a graph of CD1d+ cells (%) plotted for indicated samples.

DETAILED DESCRIPTION

The present invention provides factor VIII molecules to prevent in asubject an immune response towards said factor VIII molecules.

Factor VIII has a mature sequence made of 2332 amino acids with a domainstructure A1-a1-A2-a2-B-a3-A3-C1-C2, in which A1, A2, B, A3, C1 and C2represent domains and a1, a2 and a3 represent acidic regions linkingsaid domains. Upon proteolytic processing, which occurs after secretion,factor VIII is present in plasma under a major form consisting ofvariable length of A1-a1-A2-a2-B (the length is conditioned by thelength of the B domain) linked noncovalently to light chains consistingof A3-C1-C2.

FVIII accelerates the activation of factor X by factor IX on a suitablephospholipid surface, thereby amplifying the clotting process. Theactive form of factor VIII is made of a heterotrimer consisting inA1-a1, A2-a2 and A3-C1-C2.

In particular, the invention provides ways to prevent the expansion andfunctional activity of NKT cells. Such cells are usually classified intodistinct subsets, namely type 1 for NKT cells carrying an invariant TCRalpha chain (Valpha14 in the mouse, Valpha24 in humans), or type 2 NKTcells which present with a diverse alpha chain repertoire and is deemedto be specific for sulfatides. However, recent evidence has suggestedthat alternative subsets of NKT cells which do not fit in the type 1 ortype 2 category. It is the purpose of the present invention to includethese non conventional NKT cells, which can carry either the CD4 or theCD8 co-receptor. Upon presentation of an antigen bound to CD1d, NKTcells are rapidly activated and secrete a number of cytokines thought tobe determinant to influence other cells from both the innate andadaptive immune systems. In some circumstances, said activated NKT cellsacquire or increase cytotoxic properties.

In the context of the present invention, we made the unexpectedobservation that peptides can be presented by the CD1d molecule. Acharacteristic of the CD1d molecule is that it is made of twoanti-parallel alpha chains forming a cleft sitting atop of a platformmade of two anti-parallel beta chains. The cleft is narrow and deep andaccept only hydrophobic residues, classically deemed to be only lipids.The cleft can accommodate a sequence of 7 aminoacids characterized as ahydrophobic residue in position (P)1 and 7, and an aliphatic residue inP4. P1 is an obligate hydrophobic residue, such as F, W, H or Y.However, P7 is permissive and can contain alternative residues providedthey are not polar. Residues in P4 are preferably aliphatic but isoptional. A general sequence for a CD1d binding motif is therefore[FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] (SEQ ID NO: 7). It should however beclear for those skilled in the art that the motif is symmetrical andthat P7 can be considered as P1, and P1 can be considered as P7. Thegeneral sequence of a CD1d binding motif is provided here as a generalindication without any limiting intention.

The present invention relates to the production of factor VIII moleculesand method thereof, said molecules containing CD1d binding motif(s),which confer them with the capacity to activate NKT cells and which aremodified by elimination and/or substitution of hydrophobic residues inP1 and/or P7 with a non-hydrophobic residue, with the proviso that F inposition 309 is not substituted by S and H in position 317 is notsubstituted by A, and/or adding a non-hydrophobic residue in position P1and/or P7, with, optionally, substitution or deletion of aliphaticresidues in P4, or any combination of these, which results in a loss orsignificant reduction of the capacity of peptides or polypeptides tobind to CD and thereby results in a loss or significant reduction ofsaid peptides or polypeptides to activate NKT cells.

The present invention therefore relates to a factor VIII molecule with adomain structure A1-a1-A2-a2-B-a3-A3-C1-C2, in which A1, A2, B, A3, C1and C2 represent domains and a1, a2 and a3 acidic regions linking saiddomains said factor VIII molecule having a reduced capacity to activateNKT cells, said factor VIII molecules is obtainable by:

a. identification of at least one NKT cell epitope wherein said epitopecomprises hydrophobic aminoacid residues in position P1 and/or P7

b. modification of said epitope(s) by eliminating at least onehydrophobic aminoacid residue in position P1 and/or P7, substituting atleast one hydrophobic aminoacid residue in position P1 and/or P7 with anon-hydrophobic residue, or adding a non-hydrophobic residue in positionP1 and/or P7.

The present invention also relates to methods to obtain a factor VIIImolecule with reduced capacity to activate NKT cells comprising thesteps of:

a. identification of at least one NKT cell epitope wherein said epitopecomprises hydrophobic aminoacid residues in position P1 and/or P7

b. modification of said epitope(s) by eliminating at least onehydrophobic aminoacid residue in position P1 and/or P7, substituting atleast one hydrophobic aminoacid residue in position P1 and/or P7 with anon-hydrophobic residue or adding a non-hydrophobic residue in positionP1 and/or P7.

In a more particular embodiment, F, W, T, H or Y in positions P1 and/orP7 are replaced by a non-hydrophobic aminoacid residue, or, optionally,I, L, M or V in position P4 is replaced by a non-aliphatic residue, orany combination of these.

In yet another particular embodiment, hydrophobic residues located inposition P1 and/or P7, or, optionally, aliphatic residues located in P4,or any combination of these, are replaced by at least one non-naturalaminoacid different from non-natural F, W, T, H, Y, or by a non-aromaticorganic compound.

In yet another particular embodiment at least one aminoacid is addedwithin the CD1d binding motif, in any location within the P1 to P7sequence, which disrupts the motif, prevents its capacity to bind toCD1d and thereby its capacity to activate NKT cells.

In a preferred embodiment, non-natural aminoacids are D-aminoacids.

The present invention also relates to the factor VIII moleculescontaining CD1d binding motif(s), which confer them with the capacity ofactivate NKT cells, and which are modified by eliminating at least onehydrophobic aminoacid residue in position P1 and/or P7, substituting atleast one hydrophobic aminoacid residue in position P1 and/or P7 with anon-hydrophobic residue, or adding a non-hydrophobic residue in positionP1 and/or P7, and additionally, by deletion of aliphatic residues in P4,or any combination of these, which results in a loss or significantreduction of the capacity of peptides or polypeptides to bind to CD andthereby results in a loss or significant reduction of said peptides orpolypeptides to activate NKT cells.

Upon administration to a subject, such factor VIII molecules are notloaded on CD and thereby are prevented from activating NKT cells.

In a further aspect, the invention also covers the use of factor VIIImolecules comprising at least one substitution or deletion of F, W, T, Hor Y in positions P1 or P7 for preventing in a subject an immuneresponse towards factor VIII.

In yet a further aspect, the invention covers the use of factor VIIImolecules comprising at least one substitution or deletion of F, W, T, Hor Y in positions P1 or P7 for preventing in a subject the activation ofNKT cells towards factor VIII.

In yet a further aspect, the invention also covers the use of factorVIII molecules comprising at least one substitution or deletion of F, W,T, H or Y in positions P1 or P7 for the manufacture of a medicament forpreventing in a subject an immune response towards factor VIII.

The number of CD1d binding motifs when present in a peptide orpolypeptide, is very limited. Examples of such peptides or polypeptidesare provided below for factor VIII. Typically a polypeptide presents oneto five of these motifs.

An additional advantage of the present invention is that the CD1dmolecule presents a very limited degree of polymorphism. It is thereforeobvious for the one skilled in the art that the same aminoacidsubstitutions, addition or deletions according to the present inventionprovide peptides or polypeptides useful for all or a large majority ofsubjects. This is in sharp contrast with peptide or polypeptide motifsbinding to MHC class II molecules, wherein a large number of peptidescan be delineated which contain the appropriate sequence. This is due tothe minimum constraints imposed to MHC class II binding peptides and tothe large polymorphism of class II molecules.

Factor VIII molecules which are the object of the present invention areidentified as follows:

(1) optionally, the aminoacid sequence of factor VIII is evaluated forthe presence of at least one CD motif containing an hydrophobic residuein P1 and P7, and an aliphatic residue in P4. A general sequence such as[FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] (SEQ ID NO: 7) can be used for usingalgorithms well known in the art such ashttp://expasy.org/tools/scanprosite/

This general sequence should be considered as a tool to help identifyingwhich sequence(s) in said peptide or polypeptide contain a motif whichcould enable said peptide or polypeptide to activate NKT cells.

(2) the capacity of the peptide or polypeptide to bind to CD1d is testedin vitro using a cell line expressing the CD1d molecule. Examples ofsuch cell lines are known in the art and used to produce examples in thepresent application (for instance, JAWS2 cells and U937 cells). In apreferred embodiment, the cell line is not presenting MHC class IImolecules and is transduced for hyperexpression of CD1d using a viralvector containing the DNA sequence of CD1d or any other means known inthe art to introduce a gene in a cell. Methods for cell transduction areknown in the art. The cell line is loaded in culture with the peptide orpolypeptide, or with a synthetic peptide encompassing the correspondingsequence. Such synthetic peptides are easily produced by synthesis,using for instance the fmoc solid phase synthesis well known in the art.Efficient presentation of the peptide, polypeptide or correspondingsynthetic peptide by the CD molecule is then evaluated by measuring theactivation of NKT cells. Such cells can be obtained from peripheralblood by, for instance, magnetic sorting and maintained in culture, inthe presence of cytokines such as IL-2, IL-15 or IL-7. These methods aredescribed in the art (see for instance Godfrey et al, Nature reviewsImmunology 2010, 11: 197-206). Activation of NKT cells is assessed usingmethods such as evaluation of cytokine production.

Alternatively, peptides actually presented by antigen-presenting cellsin CD1d molecules can be eluted and separated by various chromatographymethods. Full description of such methodology will be found in Scott etal, Immunity, 12: 711-720, 2000. Said peptides are then sequenced toidentify which aminoacid residues are located in P1 and P7.

Alternatively, said synthetic peptides can be loaded on dimers,tetramers or polymers of the CD molecule to detect NKT cells specificfor such peptide. One possibility is to use fluorescence-labeledtetramers and detection using a fluorescence-activated cell sortingsystem (facs).

(3) the aminoacid sequences identified as being able to activate NKTcells and, optionally, identified by algorithms, are then modified byeither substitution or deletion. In a preferred embodiment, F, W, T, Hor Y in positions P1 and/or P7 are replaced by at least one aminoaciddifferent from F, W, T, H, Y. Natural aminoacids can be modified bypost-transcriptional modifications or substituted with chemical groupssuch as methyl groups. In another preferred embodiment, F, W, T, H or Yin positions P1 and/or P7 are replaced by any suitable alternativenon-natural aminoacid. Examples of non-natural aminoacid residues areD-aminoacids. In yet another embodiment, F, W, T, H or Y in positions P1and/or P7 are replaced by at least one aminoacid different from F, W, T,H, Y. In another preferred embodiment, F, W, T, H or Y in position P1 isreplaced by at least one aminoacid different from F, W, T, H, Y, by anysuitable alternative non-natural aminoacid or by a non-aromatic organiccompound. Such aminoacid substitution is obtained using methods wellknown in the art. In yet a further preferred embodiment, F, W, T, H or Yin position P1 is deleted. In yet another embodiment, F, W, T, H or Y inpositions P1 and P7 are deleted. Methods to carry out said deletions arewell known in the art. In yet another particular embodiment at least oneaminoacid is added within the CD1d binding motif, in any location withinthe P1 to P7 sequence.(4) optionally, it may be advantageous to replace hydrophobic aminoacidresidues adjacent to the first (P1) and or the last (P7) position bynon-hydrophobic residues. Said hydrophobic residues can be locatedwithin the flanking regions of the epitope or within the epitopesequence itself at positions P2 or P6). Positions P−2 and P−1, P2 and/orP6, P8 and P9, located at the amino-terminal end or carboxy-terminal endof the epitope, respectively, are advantageously occupied bynon-hydrophobic residues, namely aminoacids different from F, W, T, H orY, which further reduce the affinity of the epitope for CD1 d andthereby the capacity of the epitope to activated NKT cells.(5) NKT cells are then tested for their reactivity with the peptidesmodified as described in (3). Alternatively, the full-length proteinfrom which the peptide is derived can be produced with the modificationof the sequence as described in (3). Various methods can be used todetermine whether NKT cells have lost or have reduced their capacity toreact to the modified peptide or protein. These methods are known in theart. Proliferation of NKT cells can be assessed by incorporation ofradioactive thymidine, or by evaluating the concentration of cytokinesproduced in culture medium. Alternatively, NKT cells can be evaluated byELISPOT using a variety of antibodies directed towards cytokines ormolecules associated with the cytolytic properties of the cells, such asgranzyme B. Alternatively, NKT cells can be evaluated for earlysignaling events, such as phosphorylation of Fyn or surface activationmarkers.

In particular, analysis of the sequence of the A1 domain of factor VIIIhas identified two regions containing motifs enabling factor VIII tobind to the CD1d molecule, according to the methods described above.These 2 regions encompass aminoacid residues 190 to 209 and 309 to 323and preferably 309 to 316, with sequence:

190-209: QTLHKFILLFAVFDEGKSWH (SEQ ID NO: 1) 309-323 FCHISSHQHDGMEAY(SEQ ID NO: 2)

Hydrophobic aminoacid residues are underlined and each of the 2sequences contain 2 CD1d binding motifs, namely:

H193-F199

F195-V201 (P7 is permissive and can accommodate valine)

F309-H315

H317-Y323

According to the present invention medicaments are envisaged for thetreatment of diseases wherein administration of factor VIII is required,such as in congenital or acquired deficiency of factor VIII, and indisorders in which it is beneficial to administer factor VIII, such inbleeding disorders associated with acute consumption of factor VIII asobserved in septic shock, polytrauma and acute fibrinolysis, or inuncontrolled bleeding associated with cerebral hemorrhage.

The medicament of the invention is usually, though not necessarily, a(pharmaceutical) formulation comprising as active ingredient at leastone of the factor VIII molecules of the invention or a gene therapeuticvector capable of expressing said factor VIII molecules. Apart from theactive ingredient(s), such formulation will comprise at least one of a(pharmaceutically acceptable) diluent.

In general, administration of factor VIII molecules of the inventionprevents activation of the innate immune system, more particularlyactivation of NKT cells, more particularly the production of cytokinesassociated with NKT cell activation.

The identification of a NKT-cell epitope in the context of the presentinvention is known to a person skilled in the art. For instance, peptidesequences isolated from the factor VIII molecule are tested by, forexample, NKT cell biology techniques, to determine whether the peptidesequences elicit a NKT cell response. Those peptide sequences found toelicit a NKT cell response are defined as having NKT cell stimulatingactivity. Human NKT cell stimulating activity can further be tested byculturing NKT cells obtained from an individual sensitized to factorVIII, and determining whether proliferation of NKT cells occurs inresponse to the peptide/epitope as measured, e.g., by cellular uptake oftritiated thymidine. Stimulation indices for responses by NKT cells topeptides/epitopes can be calculated as the maximum CPM in response to apeptide/epitope divided by the control CPM. A NKT cell stimulation index(S.I.) equal to or greater than two times the background level isconsidered “positive.” Positive results are used to calculate the meanstimulation index for each peptide/epitope for the group ofpeptides/epitopes tested. An immunogenic peptide having a NKT cellstimulation index of greater than or equal to 2 is considered useful asa candidate to carry out the substitution or deletion of hydrophobicaminoacid residues as described in the present invention.

A peptide or polypeptide of the invention is said to be of reducedcapacity to activate NKT cells when the S.I. is less than two times thebackground level, or when levels of cytokines produced upon stimulationis less than 50% of that produced when stimulation is carried out withthe natural peptide or polypeptide sequence, or when the levels ofcytolytic agents such as granzyme B is reduced by at least twofold ascompared to peptides or polypeptides of natural sequence.

Soluble CD1d molecules are obtained and made tetrameric by synthesisand/or chemical coupling. The CD1d molecule is purified by affinitychromatography. Soluble CD1d molecules are incubated with abiotin-labeled reference peptide produced according to its strongbinding affinity for said CD1d molecule. Peptides to be assessed forCD1d binding are then incubated at different concentrations and theircapacity to displace the reference peptide from its CD1d binding site iscalculated by addition of neutravidin. Methods can be found in forinstance in Texier et al., (2000) J. Immunology 164, 3177-3184) forpeptides presented by the MHC class II determinants, but the method caneasily be applied to CD1d-restricted NKT cell epitopes.

If two or more aminoacid sequences which share an area of overlap in thenative peptide or polypeptide sequence are found to have human NKT cellstimulating activity, as determined by T cell biology techniques,mutation or deletion of hydrophobic aminoacid residues may be carriedout for residues belonging to one or to both of the sequences.

The route of administration for factor VIII molecules of the presentinvention may vary according to the indication. Examples are intravenousor subcutaneous injection of factor VIII, but the present invention alsocovers alternative routes of administration such as intranasal, oral,sublingual, percutaneous or intramuscular.

Factor VIII molecules of the present invention can be produced usingmethods know in the art for the production of recombinant proteins usingexpression systems such as bacterial cells, yeast cells, insect cells,plant cells or mammalian cells.

Factor VIII molecules of the invention can be produced by recombinantexpression in, e.g., bacterial cells (e.g. Escherichia coli), yeastcells (e.g., Pichia species, Hansenula species, Saccharomyces orSchizosaccharomyces species), insect cells (e.g. from Spodopterafrupperda or Trichoplusia ni), plant cells or mammalian cells (e.g.,CHO, COS cells). The construction of the therefore required suitableexpression vectors (including further information such as promoter andtermination sequences) involves meanwhile standard recombinant DNAtechniques. Recombinantly produced factor VIII molecules of theinvention can be derived from a larger precursor protein, e.g., viaenzymatic cleavage of enzyme cleavage sites inserted adjacent to the N-and/or C-terminus of the peptide or polypeptide, followed by suitablepurification.

The present invention also relates to nucleic acid sequences encodingthe factor VIII molecules of the present invention and methods for theiruse, e.g., for recombinant expression or in gene therapy. In particular,said nucleic acid sequences are capable of expressing factor VIIImolecules of the invention.

In gene therapy, recombinant nucleic acid molecules encoding the factorVIII molecules of the present invention can be used as naked DNA or inliposomes or other lipid systems for delivery to target cells. Othermethods for the direct transfer of plasmid DNA into cells are well knownto those skilled in the art for use in human gene therapy and involvetargeting the DNA to receptors on cells by complexing the plasmid DNA toproteins. In its simplest form, gene transfer can be performed by simplyinjecting minute amounts of DNA into the nucleus of a cell, through aprocess of microinjection. Once recombinant genes are introduced into acell, they can be recognized by the cell normal mechanisms fortranscription and translation, and a gene product will be expressed.Other methods have also been attempted for introducing DNA into largernumbers of cells. These methods include: transfection, wherein DNA isprecipitated with calcium phosphate and taken into cells by pinocytosis;electroporation, wherein cells are exposed to large voltage pulses tointroduce holes into the membrane); lipofection/liposome fusion, whereinDNA is packed into lipophilic vesicles which fuse with a target cell;and particle bombardment using DNA bound to small projectiles. Anothermethod for introducing DNA into cells is to couple the DNA to chemicallymodified proteins. Adenovirus proteins are capable of destabilizingendosomes and enhancing the uptake of DNA into cells. Mixing adenovirusto solutions containing DNA complexes, or the binding of DNA topolylysine covalently attached to adenovirus using protein crosslinkingagents substantially improves the uptake and expression of therecombinant gene. Adeno-associated virus vectors may also be used forgene delivery. As used herein, “gene transfer” means the process ofintroducing a foreign nucleic acid molecule into a cell, which iscommonly performed to enable the expression of a particular productencoded by the gene. The said product may include a protein,polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Genetransfer can be performed in cultured cells or by direct administrationinto mammals. In another embodiment, a vector comprising a nucleic acidmolecule sequence encoding a factor VIII molecule according to theinvention is provided. In particular embodiments, the vector isgenerated such that the nucleic acid molecule sequence is expressed onlyin a specific tissue. Methods of achieving tissue-specific geneexpression are well known in the art, e.g., by placing the sequenceencoding a factor VIII molecule of the invention under control of apromoter, which directs expression of said molecule specifically in oneor more tissue(s) or organ(s). Expression vectors derived from virusessuch as retroviruses, vaccinia virus, adenovirus, adeno-associatedvirus, herpes viruses, RNA viruses or bovine papilloma virus, may beused for delivery of nucleotide sequences (e.g., cDNA) encodingpeptides, homologues or derivatives thereof according to the inventioninto the targeted tissues or cell population. Methods which are wellknown to those skilled in the art can be used to construct recombinantviral vectors containing such coding sequences. Alternatively,engineered cells containing a nucleic acid molecule coding for a peptideor polypeptide according to the invention may be used in gene therapy.

Factor VIII of the present invention can be modified by methods known inthe art.

Factor VIII molecules can encompass the entire aminoacid sequence offactor VIII or only parts of it. One example is provided by B domaindeleted factor VIII molecules. The B domain is dispensable for thefunction of factor VIII and can therefore be deleted without affectingfactor VIII function. The a2 and a3 domains are usually linked byaminoacid sequence which can be artificial or can represent sequencesfrom the B domain itself. Factor VIII molecules can be modified byaddition and/or substitution of aminoacids so as to increase factor VIIIstability by, for instance, reducing the decoy rate of the A2 domain, orby increasing factor VIII resistance to proteolytic enzymes. Factor VIIImolecules can be stabilized by coupling to Fcgamma part of antibodies,for instance to increase its recycling trough the FcRn receptor. FactorVIII can be stabilized by reducing its degradation usingpolyethyleneglycol residues (PEG derivatives) or alternative chemicalsubstitution. All these modifications are considered as within the scopeof the present invention.

The prior art (Swaroop et al., The Journal of Biological Chemistry, vol272, pp 24121-24124, 1997; Cerullo et al., Molecular Therapy, vol 15, pp2080-2087, 2007) has identified that mutation of F309 (SEQ ID NO: 2)increases the rate of production of factor VIII molecules by cellstransfected with a factor VIII construct. Cerullo et al. observed anon-significant reduction of the production of inhibitor antibodies infactor VIII KO mice treated with the F309 mutant, teaching away the oneskilled in the art from the use of such mutant for reducing factor VIIIimmunogenicity. Moreover, in said prior art, there is no mention thatF309 could be part of a CD1d binding motif.

The medicament of the invention is usually, but not necessarily a(pharmaceutical) formulation comprising as active ingredient at leastone factor VIII molecule of the invention, a gene therapeutic vectorcapable of expressing said factor VIII molecule. Apart from the activeingredient(s), such formulation will comprise at least one of a(pharmaceutically acceptable) diluent. Typically, pharmaceuticallyacceptable compounds can be found in, e.g., a Pharmacopeia handbook(e.g. US-, European- or International Pharmacopeia). The medicament orpharmaceutical composition of the invention normally comprises a(prophylactically or therapeutically) effective amount of the activeingredient(s) wherein the effectiveness is relative to the condition ordisorder to be prevented or treated.

The medicament or pharmaceutical composition of the invention may, in apreferred embodiment, need to be administered to a subject in need aspart of a prophylactic or therapeutic regimen comprising multipleadministrations of said medicament or composition. Said multipleadministrations usually occur sequentially and the time-interval betweentwo administrations can vary and will be adjusted to the nature of theactive ingredient and the nature of the condition to be prevented ortreated. The amount of active ingredient given to a subject in need of asingle administration can also vary and will depend on factors such asthe physical status of the subject (as for instance weight and age), thestatus of the condition to be prevented or treated, and the experienceof the treating doctor, physician or nurse.

The term “diluents” refers for instance to physiological salinesolutions. The term “pharmaceutically acceptable carrier” means anymaterial or substance with which the active ingredient is formulated inorder to facilitate its application or dissemination to the locus to betreated, for instance by dissolving, dispersing or diffusing the saidcomposition, and/or to facilitate its storage, transport or handlingwithout impairing its effectiveness. They include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents (forexample phenol, sorbic acid, chlorobutanol), isotonic agents (such assugars or sodium chloride) and the like. Additional ingredients may beincluded in order to control the duration of action of the activeingredient in the composition. The pharmaceutically acceptable carriermay be a solid or a liquid or a gas which has been compressed to form aliquid, i.e. the compositions of this invention can suitably be used asconcentrates, emulsions, solutions, granulates, dusts, sprays, aerosols,suspensions, ointments, creams, tablets, pellets or powders. Suitablepharmaceutical carriers for use in said pharmaceutical compositions andtheir formulation are well known to those skilled in the art, and thereis no particular restriction to their selection within the presentinvention. They may also include additives such as wetting agents,dispersing agents, stickers, adhesives, emulsifying agents, solvents,coatings, antibacterial and antifungal agents (for example phenol,sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodiumchloride) and the like, provided the same are consistent withpharmaceutical practice, i.e. carriers and additives which do not createpermanent damage to mammals. The pharmaceutical compositions of thepresent invention may be prepared in any known manner, for instance byhomogeneously mixing, coating and/or grinding the active ingredients, ina one-step or multi-steps procedure, with the selected carrier materialand, where appropriate, the other additives such as surface-activeagents. They may also be prepared by micronisation, for instance in viewto obtain them in the form of microspheres usually having a diameter ofabout 1 to 10 μm, namely for the manufacture of microcapsules forcontrolled or sustained release of the active ingredients.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.

The following examples are provided here to illustrate the invention.There is however no intention to limit the invention to such examples.

Example 1

Binding of Peptide of SEQ ID NO: 1 to CD1d Tetramers

A peptide encompassing SEQ ID NO: 1 was obtained by chemical synthesis.This peptide was used to load CD1d tetramers (ProImmune) by overnightincubation.

NKT cells were prepared from the spleen of naïve mice by depletion ofall CD4(−) cells using magnetic beads.

CD4+ cells were then incubated with CD1d tetramers loaded by peptide ofSEQ ID NO: 1 (P28 WT in FIG. 1), which corresponds to aminoacid sequence190 to 209.

FIG. 1 shows that ±1% of CD4+ T cells were detected. Further, peptide ofSEQ ID NO: 1 was separated into 2 parts and synthetic peptides coveringthe sequence 190-200 (SEQ ID NO: 3, P28 A in FIG. 1) and 200-210 (SEQ IDNO: 4, P28 B in FIG. 1) were produced.

Sequence of peptide of SEQ ID NO: 3 is:

QTLHKFILLFA (corresponding to aminoacid sequence 190 to 200)

Sequence of peptide of SEQ ID NO: 4 is:

AVFDEGKSWHS (corresponding to aminoacid sequence 200 to 210)

The Figure indicates that a major CD1d binding motif is located in SEQID NO: 3, and limited though significant % of NKT cells were detected bythe peptide of SEQ ID NO: 4.

It was therefore concluded that a major CD binding motif was located inpeptide of SEQ ID NO: 3.

Example 2

Activation of NKT Cells by Immunization with JAWS2 Cells Loaded withPeptide of SEQ ID NO: 1

JAWS2 cells express CD1d but not MHC class II determinants. These cellswere used to show that the presentation of peptide of SEQ ID NO: 1 canoccur via CD1d.

JAWS2 cells were incubated 2 h at 37° C. with peptide of SEQ ID NO: 1(10 μg/ml) and thoroughly washed twice. Cells were then treated withmitomycin to block cell division and washed 4 times to eliminatemitomycin. A cell suspension containing 2×10⁶ cells in physiologicalserum was injected by the intraperitoneal route in each of a series of 3naïve factor VIII KO mice. As a control, 3 naïve factor VIII KO micereceived the same number of JAWS2 cells which had not been incubatedwith peptide of SEQ ID NO: 1.

Five days after IP injection, the mice were sacrificed and their spleenprepared as above using magnetic beads to eliminate all CD4(−) cells.

CD4+ cells were then incubated with tetramers loaded with peptide of SEQID NO: 1.

FIG. 2 shows that a significant proportion of NKT cells (8%) present inthe natural repertoire are specific for peptide of SEQ ID NO: 1 and thatsuch proportion is doubled (16%) when peptide of SEQ ID NO: 1 ispresented by JAWS2 cells.

The specificity of NKT cell detection is shown by the lack of NKTdetection with unloaded tetramers as well as when tetramers were loadedwith a peptide unrelated to peptide of SEQ ID NO: 1.

It was therefore concluded that presentation of peptide of SEQ ID NO: 1occurred via CD1d presentation and that this presentation was sufficientas to bind and activate NKT cells.

Example 3

Administration of Mutated Factor VIII Reduces the Formation ofAntibodies to Factor VIII

To determine whether mutation aimed at eliminating CD1d binding motifsin factor VIII could reduce the concentration of anti-factor VIIIantibodies, wildtype or mutated factor VIII were cloned in the plasmidpGC5AM-EN. Mutations in positions F309 and H315, each time into aalanine, were introduced in the sequence of factor VIII using PCR andthe splicing by overlap extension (SOE-PCR) technology.

The plasmids containing either the native or the mutated sequence werechecked by sequencing and prepared by maxiprep.

Plasmids (100 μg in 2 ml) were directly administered into naïve factorVIII KO C57BL/6 mice (3 mice per group) using the hydrodynamic pressuremethod (injection made in less than 7 seconds), known in the art todirect plasmids essentially to the liver. Control mice were injectedusing the same method but with naked plasmid (n=3).

A total of 3 injections were made at intervals of 10 days.

Ten days after the last injection, plasma concentrations of antibodiesto factor VIII and the concentration of antibodies inhibiting thefunction of factor VIII were measured by ELISA and chromogenic assay,respectively.

FIG. 3A indicates that a 3.6-fold reduction of antibodies was observedin mice injected with the factor VIII mutant F309A-H315A. FIG. 3B showsa 3.8-fold reduction of antibodies inhibiting the function of factorVIII.

These data showed that elimination of a single CD1d binding motif in thefactor VIII molecule is sufficient to significanity reduced theconcentration of specific antibodies, including those inhibiting thefunction of factor VIII.

Example 4

Increased Expression of CD1d on Human Antigen-Presenting Cells UponIncubation with a Peptide Containing a CD1d-Binding Motif

To determine whether human antigen-presenting cells had the capacity toprocess and present a peptide epitope in the context of the CD1dmolecule, the human macrophage cell line U937 was used. Such cells donot express class II major histocompatiblity complexes and are used toassess presentation by CD1d.

The percentage of resting U937 cells expressing CD1d is low as detectedwith a specific anti-CD1d antibody. It was reasoned that, if U937 cellswere incubated with a peptide having the sequence motif enabling it tobind to CD1d, then this should be detectable by increased expression ofCD1d at cell surface. As observed for peptides encompassing MHC class IIepitopes, the binding of a peptide stabilizes the conformation of themolecule (class II, or CD1d in the present case), allowing the complexto be anchored at cell surface and reducing its intracellular recycling.

U937 cells (7×10⁵ cells) were incubated for 24h at 37° C. with 5 μg ofpeptide encompassing CD1d binding motifs or controls. The cells werethen washed and incubated with fluorescence-labeled antibodies specificto CD1d and the number of positive cells evaluated using a FACS system.

FIG. 4 shows that the percentage of cells expressing CD1d increases from6% in control experiments to 13% when peptides containing CD1d-bindingsequences were used. Data shown as histograms are means of triplicatemeasurements, with asterisk identifying results significantly higherthan control values (p<0.05).

Controls include (from left to right) unloaded U937, cells incubatedwith alpha-gal-ceramide and a conventional class II-restricted epitope(MHCII-MOG). The experiment includes sequences of factor VIII of eitherhuman (SEQ ID NO: 5) or mouse (SEQ ID NO: 6) origin corresponding toaminoacids 188-204 of the factor VIII sequence.

It is therefore concluded that human antigen-presenting cells stabilizecomplexes of peptides and CD1d at their surface when incubated with saidpeptides.

Example 5

Human Peripheral Blood Mononucleated Cells (PBMC) Contain NKT CellsSpecific for CD1d-Bound Peptidic Epitopes

In order to establish whether human PBMC contained cells reacting with aCD1d-binding peptide epitope, invariant NKT (iNKT) cells were preparedfrom a buffy coat using magnetic beads coated with an antibody specificto the Valpha24-Jalpha18 chain of the T cell receptor (so-calledinvariant chain).

Said human iNKT cells (10⁵/condition) were then incubated withmitomycine-C treated U937 cells (Ratio 1:1) loaded with 5 μg of eitherpeptide encompassing aminoacid residues 188-204 of human factor VIII(SEQ ID NO: 5) or with control peptide (MHCII-MOG). Incubation wascarried out for 2 weeks at 37° C. in RPMI containing 10% of fetal calfserum and 50 U/ml of human recombinant IL-2.

Culture plates were examined by eye for the presence of cell clusters.It is observed that clumps of cells form with U937 loaded with peptideof SEQ ID NO: 5 to the same extent that with alpha-gal-ceramide, but notwith control peptide (MHCII-MOG). FIG. 5 illustrates the average ofthree microscopic field count of this observation (minimum 10cells/cluster).

It is therefore concluded that human peripheral cell repertoire containscells defined as invariant chain NKT cells reacting with a peptidepresented by CD1d.

Example 6

Flanking Residues Located Outside of the CD Binding Cleft are Importantfor Efficient Cell Surface Presentation

The experiment shown in Example 1 indicates that a peptide of SEQ ID NO:3, corresponding to human factor VIII aminoacids 190-200, binds to CDtetramers and allows the detection of NKT cells, indicating that thissequence contained the minimum binding motif for CD1d. To determinewhether this motif was sufficient for efficient presentation by cells,the experiment reported in example 4 was repeated with peptide of SEQ IDNO: 3.

FIG. 6 shows that peptide of SEQ ID NO: 3 did not increase expression ofCD1d at the surface of U937 cells, in contrast to peptides of SEQ ID NO:5 shown in FIG. 4.

It is therefore concluded that an efficient in vivo loading of a peptidecontaining a CD1d-binding motif requires the presence of flankingresidues located outside of the CD1d cleft.

Example 7

NKT Cells Specific for CD1d-Binding Factor VIII Epitope Belong to Boththe CD4+ and CD8+ Lineages

To determine the cell lineage to which NKT cells belonged, an experimentwas performed as described in Example 2. Thus, JAWS2 cells were loadedwith either peptide of SEQ ID NO: 3, B-domain-deleted factor VIII,alpha-gal-ceramide or kept unloaded before intraperitoneal (IP)injection in factor VIII KO mice (2×10⁶ cells per mouse). Five daysafter intraperitoneal injections, the mice were sacrificed and thesplenocytes prepared. Cell suspensions were then reacted with CD1dtetramers unloaded or loaded with peptide of SEQ ID NO: 3 or withalpha-gal-ceramide.

For faccs analysis, cells binding CD1d tetramers were labeled with DX5-and CD160-specific antibodies and either anti-CD4 or anti-CD8antibodies. The table summarizes the results.

Number of DX5/CD160 cells (×10³) Tetramers CD4+ CD8+ 1 JAWS2 (—) (—) 0.30.1 2 Alpha-gal-cer 1.2 1.3 3 Peptide SEQ ID 39 3.5 NO: 3 4 JAWS2Peptide SEQ ID (—) 0.2 0.1 NO: 3 5 Alpha-gal-cer 2.2 0.8 6 Peptide SEQID 47 4.1 NO: 3 7 JAWS2 Factor VIII (—) 0.1 0.5 8 Alpha-gal-cer 1.9 1.49 Peptide SEQ ID 62 11 NO: 3 10 JAWS2 Alpha-gal-cer (—) 0.1 0 11Alpha-gal-cer 2.3 1.8 12 Peptide SEQ ID 46 1.9 NO:3

These experiments show that

-   -   the natural repertoire of NKT cells contains a significant        number of cells reacting to peptide of SEQ ID NO: 3 in both the        CD4+ and CD8+ lineages (line 3)    -   this population of cells can be further expanded by immunization        with peptide of SEQ ID NO: 3, again in both CD4+ and CD8+        lineages (line 6)    -   immunization with factor VIII is even more efficient in        expanding these cell populations (line 9)    -   immunization with alpha-gal-ceramide slightly increases the        number of CD4+ T cells specific for peptide of SEQ ID NO: 3, but        not the number of CD8+ T cells

Taken together, these results indicate that immunization with factorVIII elicits an expansion of cells belonging to the NKT lineage of boththe CD4+ and CD8+ phenotype.

Sequences SEQ ID NO: 1 QTLHKFILLFAVFDEGKSWH (human 190-209) SEQ ID NO: 2FCHISSHQHDGMEAY (human 309-323) SEQ ID NO: 3 QTLHKFILLFA (human 190-200)SEQ ID NO: 4 AVFDEGKSWHS (human 200-210) SEQ ID NO: 5KTQTLHKFILLFAVFDE (human 188-204) SEQ ID NO: 6RTQMLYQFVLLFAVFDE (mouse 188-204)

The invention claimed is:
 1. A method of treating septic shock, acutefibrinolysis, polytrauma, cerebral haemorrhage, or other bleedingdisorder, the method comprising administering to a patient in needthereof a factor VIII molecule having a reduced capacity to activate NKTcells and obtained by: (a) identifying at least one NKT cell epitopecomprising SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:5 in a factor VIIImolecule, wherein said epitope comprises a seven amino acid residuesequence (positions P1-P7) being [FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] (SEQID NO: 7); and wherein each of X₂, X₃, X₅, and X₆ can be any amino acid;and (b) modifying at least one identified NKT cell epitope bysubstituting the amino acid residue in position P1 with a residue otherthan F, W, T, H or Y, and by substituting the amino acid residue inposition P7 with a residue other than F, W, T, H or Y.
 2. The method ofclaim 1, wherein the factor VIII molecule has a domain structureA1-a1-A2-a2-B-a3-A3-C1-C2, or wherein the Factor VIII is present inplasma with a major form consisting of A1-a1-A2-a2-B linkednoncovalently to light chains consisting of A3-C1-C2, in which A1, A2,B, A3, C1 and C2 represent domains and a1, a2 and a3 represent acidicregions linking said domains.
 3. The method of claim 1, wherein thefactor VIII molecule is obtained by the further step of selecting amodified Factor VIII molecule causing a level of cytokine or ofcytolytic agents production upon stimulation of NKT cells of less than50% of the natural sequence.
 4. The method of claim 1, wherein themethod is for treating congenital or acquired haemophilia A.